Brucella is a Gram-negative intracellular bacterium whose pathogenesis is not understood. The recently sequenced genome of Brucella melitensis has identified four quorum sensing luxR homologues. LuxR transcriptional regulators have been shown to activate virulence gene expression in a number of bacteria. Our proposed goal is to identify genes associated with LuxR regulation and determine the relationship of quorum sensing to putative virulence gene expression in B. melitensis. Our specific aims are to: 1) create mutants in the four different luxR regulator gene homologs in Brucella melitensis 16M via allelic replacement; 2) using our luxR mutants, determine the extent these genes are involved in survival in macrophages and mice; 3) identify Brucella melitensis 16M genes differentially expressed during macrophage infection by the putative LuxR regulators using DNA microarrays. Thus, we will relate temporal expression of quorum sensing genes to potential virulence gene regulation and create opportunities to identify new vaccine targets and antibacterial agents.